Contents lists available at ScienceDirect Oral Oncology journal homepage: www.elsevier.com/locate/oraloncology Proteome analysis reveals that de novo regenerated mucosa over fibula flap- reconstructed mandibles resembles mature keratinized oral mucosa ☆ Vinay V. Kumar a,b,c,d , Bonney L. James a , Manuela Ruß d , Stefan Mikkat d,e , Amritha Suresh a , Peer W. Kämmerer c , Michael O. Glocker d, ⁎ a Integrated Head and Neck Oncology Research Program, Mazumdar Shaw Centre for Translational Research, Mazumdar Shaw Medical Foundation, Bommasandra Indl Area, Bangalore, India b Department of Head and Neck Surgery, Mazumdar Shaw Medical Center, Narayana Health, Bommasandra, Bengaluru, India c Department of Oral Maxillofacial and Plastic Surgery, University Medicine Rostock, Rostock, Germany d Proteome Center Rostock, University Medicine Rostock, Rostock, Germany e Core Facility Proteome Analysis, University Medicine Rostock, Rostock, Germany ARTICLE INFO Keywords: Oral cancer Free fibula flap Mandible reconstruction Keratinized mucosa Cranio-Maxillofacial Surgery Proteome signature Proteome research Regeneration Cornification Cornified cell envelope ABSTRACT Aim: The aim of this study was to determine whether intra-oral de novo regenerated mucosa (D) that grew over free fibula flap reconstructed-mandibles resembled the donor tissue i.e. external skin (S) of the lateral leg, or the recipient site tissue, i.e. keratinized oral mucosa (K). Materials and methods: Differential proteome analysis was performed with ten tissue samples from each of the three groups: de novo regenerated mucosa (D), external skin (S), and keratinized oral mucosa (K). Expression differences of cornulin and involucrin were validated by Western blot analysis and their spatial distributions in the respective tissues were ascertained by immunohistochemistry. Results: From all three investigated tissue types a total of 1188 proteins were identified, 930 of which were reproducibly and robustly quantified by proteome analysis. The best differentiating proteins were assembled in an oral mucosa proteome signature that encompasses 56 differentially expressed proteins. Principal component analysis of both, the 930 quantifiable proteins and the 56 oral mucosa signature proteins revealed that the de novo regenerated mucosa resembles keratinized oral mucosa much closer than extra-oral skin. Differentially expressed cornification-related proteins comprise proteins from all subclasses of the cornified cell envelope. Prominently expressed in intra-oral mucosa tissues were (i) cornifin-A, cornifin-B, SPRR3, and involucrin from the cornified-cell-envelope precursor group, (ii) S100A9, S100A8 and S100A2 from the S100 group, and (iii) cornulin which belongs to the fused-gene-protein group. Conclusion: According to its proteome signature de novo regenerated mucosa over the free fibula flap not only presents a passive structural surface layer but has adopted active tissue function. Introduction Attached gingiva and fixed keratinized alveolar mucosa are anato- mically peculiar entities that are only seen around the alveolar process of dentulous patients and on the crestal ridges of edentulous patients respectively. In patients who need to undergo segmental resection of the jaws for the treatment of pathologies, composite reconstruction is frequently performed with the free fibula flap. Mandibular bony defects are reconstructed using the fibula bone, external skin is replaced with the lateral leg skin, and dental rehabilitation is provided with implant- supported prosthesis [1–3]. Although the lateral leg skin paddle is also used as an intra oral lining, it is not an ideal substitute for keratinized mucosa in reconstructed jaws. Lack of adequate keratinized mucosa around dental implants in reconstructed jaws are associated with in- creased plaque accumulation, mucosal inflammation, attachment loss, and recession [4,5]. https://doi.org/10.1016/j.oraloncology.2018.01.017 Received 11 December 2017; Received in revised form 15 January 2018; Accepted 23 January 2018 ☆ Devoted to Prof. Przybylski on the occasion of his 70th birthday. ⁎ Corresponding author at: Proteome Center Rostock, University Rostock Medical Center and Natural Science Faculty, University of Rostock, Schillingallee 69, 18059 Rostock, Germany. E-mail address: michael.glocker@med.uni-rostock.de (M.O. Glocker). URL: http://www.pzr.med.uni-rostock.de (M.O. Glocker). Abbreviations: D, de novo regenerated mucosa over free fibula flap; K, keratinized oral mucosa; S, skin over the lateral leg; CE, cornified cell envelope; DGER, denture-guided epithelial regeneration Oral Oncology 78 (2018) 207–215 Available online 20 February 2018 1368-8375/ © 2018 Elsevier Ltd. All rights reserved. T